Several publications are referenced in this application by author names and year of publication in parenthesis in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
Genetic engineering of plants involves the development and application of technology for genetic transformation through the direct manipulation of the plant genome and plant gene expression by the introduction of novel DNA. One method of transformation employs a derivative of the tumor inducing (Ti) plasmid from the bacterium, Agrobacterium tumefaciens. Other methods utilize direct gene transfer into protoplasts using biolistics, electroporation, polyethylene glycol treatment.
While the incorporation of transforming DNA in the nucleus of plant cells is well known to those skilled in the art, transformation protocols that selectively identify transformed plastid DNA, to the exclusion of other genetic compartments have not yet been described. With the above-described methods, if plastid transformation only is desired, nuclear transformants may express the gene encoding the selectable marker and result in the generation of false positives.
The need for plastid-specific marker genes is based on this observation. In earlier work, selection for kanamycin resistance of pTNH32-bombarded tobacco leaves yielded a large number of nuclear transformants (Carrer et al., 1993). Indeed, recovery of nuclear gene transformants with other plastid kan genes (Cornelissen and Vandewiele 1989), and with promoterless kan constructs (Koncz et al. 1989) confirms that kanamycin resistant clones may be readily obtained by transformation with constructs that were not designed for expression in the nucleus. Additionally, nuclear gene transformants in tobacco may also be recovered by selection for spectinomycin resistance genes designed for expression in plastids.
Given the large number of plastid genomes in plant cells, the ability to select for the transformed genome in culture is a key element in achieving successful transformation. Selection markers have been identified by screening cultured plant cells for mutants resistant to various substances, such as antibiotics and herbicides. Such antibiotics and herbicides are listed in Table I, below. However, to date, a method has not been developed that will facilitate plastid transformation with the concomitant exclusion of the selection of nuclear transformants. The development of such a system minimizes the false positives that result when a nuclear transformation event occurs.
RNA editing is a process that post-transcriptionally alters RNA sequences. Until recently, it was believed that chloroplasts, in contrast to mitochondria, did not utilize RNA editing and that the prediction of amino acid sequences from the corresponding gene sequences was generally correct. While most chloroplast genes begin with the canonical ATG start codon, genes have been identified that encode an ACG at a position that corresponds to the 5' terminal ATG in homologous genes in other species. Recently it has been shown that this ACG codon is not conserved at the mRNA level. It is converted to a functional AUG codon by C to U editing (Hoch et al., 1991). Most of the edited codons found to date, restore amino acids that are conserved in the corresponding peptides from chloroplasts of other species. This editing process is plastid specific. Genes edited in the plastid are not edited in the nucleus or other organelles of the plant. The present invention provides DNA constructs and methods to facilitate the selection of stably transformed plastids, based upon a requirement for RNA editing in the transforming constructs which occurs exclusively in the plastid. Targeted manipulation of the plastid genome can now be performed with greater ease. Such manipulations include gene replacement, gene deletion, insertion of foreign genes and expression of recombinant proteins in plastids.